ER-to-lysosome-associated degradation acts as failsafe mechanism upon ERAD dysfunction

The endoplasmic reticulum (ER) produces proteins destined to organelles of the endocytic and secretory pathways, the plasma membrane, and the extracellular space. While native proteins are transported to their intra- or extracellular site of activity, folding-defective polypeptides are retro-translocated across the ER membrane into the cytoplasm, poly-ubiquitylated and degraded by 26 S proteasomes in a process called ER-associated degradation (ERAD). Large misfolded polypeptides, such as polymers of alpha1 antitrypsin Z (ATZ) or mutant procollagens, fail to be dislocated across the ER membrane and instead enter ER-to-lysosome-associated degradation (ERLAD) pathways. Here, we show that pharmacological or genetic inhibition of ERAD components, such as the α1,2-mannosidase EDEM1 or the OS9 ERAD lectins triggers the delivery of the canonical ERAD clients Null Hong Kong (NHK) and BACE457Δ to degradative endolysosomes under control of the ER-phagy receptor FAM134B and the LC3 lipidation machinery. Our results reveal that ERAD dysfunction is compensated by the activation of FAM134B-driven ERLAD pathways that ensure efficient lysosomal clearance of orphan ERAD clients.


Introduction
Maintenance of cellular homeostasis relies on efficient clearance of folding-defective gene products.ERAD, for ER-associated protein degradation, is an acronym coined to describe the proteasomal clearance from the ER of misfolded pro-alpha factor in a reconstituted yeast system (McCracken and Brodsky, 1996).ERAD now defines all the pathways in eukaryotic cells that ensure recognition of folding-defective polypeptides in the ER lumen or membrane and control their transport into the cytosol for poly-ubiquitylation and degradation by 26 S proteasomes (Fig. 1A) (Christianson et al, 2023;Hebert et al, 2010;Sun and Brodsky, 2019).
Recently, it has been observed that large misfolded proteins fail to be transported across the ER membrane into the cytosol and fail therefore to enter ERAD pathways.These polypeptides are segregated in specialized ER subdomains and are eventually delivered to degradative endolysosomes or autolysosomes (as defined in (Bright et al, 2016;Huotari and Helenius, 2011) and (Klionsky et al, 2014), respectively, Fig. 1B) in Metazoan cells, or to vacuoles in yeast and plant cells (Rudinskiy and Molinari, 2023).The term ERLAD for ER-to-lysosome-associated degradation was coined to describe the delivery from the ER to degradative RAB7/ LAMP1-positive endolysosomes of disease-causing ATZ polymers (Fregno et al, 2018;Fregno et al, 2021) or of misfolded procollagen (Forrester et al, 2019) (Fig. 1B, arrows 1 and 2, respectively).Nowadays, ERLAD is an umbrella term that embraces all the autophagic and non-autophagic pathways that deliver ERADresistant misfolded polypeptides from the ER to degradative endolysosomes/autolysosomes/vacuoles (Fig. 1B) (Fregno and Molinari, 2019;Gubas and Dikic, 2022;Klionsky et al, 2021;Rudinskiy and Molinari, 2023).Mechanistically, the clearance from the ER of ATZ polymers and of procollagen molecules relies on cycles of de-/re-glucosylation of N-linked oligosaccharides that prolong calnexin (CNX) binding to favor engagement of the ER membrane protein FAM134B (Fregno et al, 2018(Fregno et al, , 2021)).FAM134B has been first described as a LC3-binding protein, whose role in autophagic clearance of ER subdomains is activated upon nutrient restriction (Khaminets et al, 2015).More recently, our group established that its role as an ER-phagy receptor (i.e., a protein that controls lysosomal clearance of ER portions and content) is also activated by the luminal accumulation of ERAD-resistant misfolded polypeptides (Forrester et al, 2019;Fregno et al, 2018Fregno et al, , 2021)).Disposal of ATZ monomers proceeds via ERAD.In contrast, clearance from cells of ATZ polymers relies on their delivery into lysosomal degradative compartments and involves ATG5 and ATG7 autophagy gene products that control LC3 lipidation (Chu et al, 2014;Fregno et al, 2018;Hidvegi et al, 2010;Kroeger et al, 2009;Pastore et al, 2013;Sun et al, 2023;Teckman and Perlmutter, 2000).Despite the intervention of the LC3 lipidation machinery, in all mammalian cell lines tested in our lab, the lysosomal clearance of ATZ polymers does not require the intervention of autophagosomes.Rather, it relies on SNARE proteins-driven fusion of ER subdomains or vesicles containing ATZ polymers with RAB7/ LAMP1-positive endolysosomes (Fig. 1B, arrow 1) (Fregno et al, 2018(Fregno et al, , 2021)).In contrast, autophagosomes are involved in disposal of procollagen molecules, which proceeds via macro-ER-phagy (Fig. 1B, arrow 2) (Forrester et al, 2019).A variety of ERLAD clients is found in the literature, but for few of them the degradative mechanisms have been characterized in molecular detail (Rudinskiy and Molinari, 2023).Certainly, a variety of ERLAD pathways does operate in eukaryotic cells.These catabolic programs are regulated by client-and tissue-specific ER-phagy receptors, whose activity in promoting clearance of ER subdomains is activated by the intraluminal or membrane accumulation of misfolded polypeptides, including FAM134B (Forrester et al, 2019;Fregno et al, 2018;Fregno et al, 2021), CCPG1 (Ishii et al, 2023;Smith et al, 2018) and FAM134B-2 (Kohno et al, 2019).Their activation ensures delivery of ER content to be removed from cells to degradative organelles via LC3-dependent delivery (Fig. 1B, arrow 1), macro-ER-phagy (arrow 2), or micro-ER-phagy (arrow 3).
Notably, ERAD inhibition delays, rather than blocks, degradation of ERAD clients, hinting at alternative pathways intervening to ensure efficient clearance of misfolded proteins generated in the ER under conditions of ERAD impairment or overload (Molinari, 2007).To uncover a possible interplay of proteasomal (ERAD) and lysosomal (ERLAD) quality control of defective gene products synthesized in the ER, we assessed the capacity of the ERLAD machinery to support clearance of Null Hong Kong (NHK) and BACE457Δ, two classical ERAD clients, upon pharmacologic or genetic ERAD inactivation.The NHK variant of alpha1 antitrypsin is a disease-causing folding-defective glycoprotein (Sifers et al, 1988).BACE457Δ is a folding-defective splice variant of betasecretase (Molinari et al, 2002).Characterization of the machineries ensuring proteasomal clearance of NHK and BACE457Δ from the mammalian ER revealed general principles of ERAD: (i) the role of mannose processing by EDEM proteins to extract terminally misfolded polypeptides from the CNX folding cycle (Chiritoiu et al, 2020;Hirao et al, 2006;Liu et al, 1997Liu et al, , 1999;;Molinari et al, 2003;Oda et al, 2003;Olivari et al, 2006); (ii) the engagement of OS9 ERAD lectins that deliver misfolded polypeptides to various clientspecific dislocons embedded in the ER membrane that control ERAD clients retro-translocation into the cytosol for polyubiquitylation and proteasomal degradation (Fig. 1A) (Bernasconi et al, 2010a;Bernasconi et al, 2008;Christianson et al, 2008;Ninagawa et al, 2011;Sugimoto et al, 2017)); (iii) the involvement of members of the protein disulfide isomerase (Guerra et al, 2018;Molinari et al, 2002;Ushioda et al, 2008) and of the peptidyl-prolyl cis/trans isomerase superfamilies (Bernasconi et al, 2010b).As such, NHK and BACE457Δ are among the best-characterized clients of the ERAD machinery.Here, we monitor their fate in cells with pharmacologically-or genetically induced dysfunction of ERAD.Our data reveal the intervention of FAM134B-driven ERLAD pathways to warrant efficient clearance of misfolded ERAD clients in cells characterized by dysfunctional ERAD.

The ERAD client NHK is normally not delivered to endolysosomes for clearance
The canonical ERLAD client ATZ is delivered to LAMP1-positive endolysosomes for clearance (Fregno et al, 2018(Fregno et al, , 2021)).Consistently, when mouse 3T3 cells expressing HA-tagged ATZ were incubated with BafA1 to inhibit lysosomal hydrolases and preserve delivered material in the lumen of degradative organelles (Klionsky et al, 2008), ATZ accumulates in endolysosomes that display LAMP1 at the limiting membrane as seen in Confocal laser scanning microscopy (CLSM) (Fig. 2E).ATZ delivery to the degradative compartment is quantified with LysoQuant (Morone et al, 2020), a deep-learning-based analysis software for segmentation and classification of fluorescence images (Fig. 2I,  ATZ+BafA1).The ERAD client NHK, whose clearance from cells is not delayed upon inhibition of lysosomal enzymes with BafA1 (Fig. 2D) (Liu et al, 1999) is poorly delivered within LAMP1-positive degradative compartments (Fig. 2F,J and quantification in Fig. 2I,O, NHK+BafA1).

Pharmacologic inhibition of ERAD triggers lysosomal delivery of NHK
Motivated by the results of the biochemical analyses showing that inactivation of lysosomal hydrolases stabilizes NHK when ERAD is inactive (Fig. 2A-C), we examined the delivery to endolysosomes of NHK upon pharmacologic inhibition of the ERAD machinery in mouse 3T3 cells.CLSM reveals the enhanced accumulation of NHK in endolysosomes that display LAMP1 at the limiting membrane in cells where ERAD has been inhibited with KIF (Fig. 2G,I), or with PS341 (Fig. 2H,I).Thus, inhibition of ERAD at early (KIF) and late steps (PS341) of client selection, activates channeling of misfolded polypeptides into ERLAD pathways that deliver them to degradative LAMP1-positive compartments.The degradative nature of the LAMP1-positive endolysosomes where NHK accumulates in cells treated with BafA1 (Fig. 2F-I,K,M,O) is confirmed by the disappearance of the NHK immunoreactivity from the endolysosomal lumen upon BafA1 washout (Fig. 2L,N,O) and (Fregno et al, 2018).

Genetic inhibition of ERAD upon silencing of EDEM1 triggers delivery of NHK to degradative endolysosomes
EDEM1 is an active α1,2-mannosidase that ensures the extraction of terminally misfolded glycoproteins from the CNX chaperone system to direct them for proteasomal degradation (Molinari et al, 2003;Oda et al, 2003;Olivari et al, 2006;Olivari and Molinari, 2007).Silencing of EDEM1 expression delays ERAD of glycoproteins (Molinari et al, 2003).To verify whether silencing of EDEM1 expression diverts NHK into the ERLAD pathway to compensate for ERAD dysfunction, we compared delivery of HA-tagged NHK in the LAMP1-positive endolysosomes in HEK293 cells expressing a scrambled short hairpin RNA (shCTRL), or a short hairpin RNA targeting the EDEM1 sequence (shEDEM, Fig. 3A).Cells were exposed to BafA1 to preserve the NHK fraction possibly delivered in the lumen of endolysosomes.CLSM reveals the enhanced accumulation of NHK in endolysosomes that display LAMP1 at the limiting membrane in cells with reduced EDEM1 levels (Fig. 3B, lower panels), which has been quantified by LysoQuant (Fig. 3C).

FAM134B drives lysosomal delivery of NHK upon ERAD inhibition
Misfolded polypeptides that fail to engage the ERAD machinery (ATZ polymers are shown as examples in Fig. 2E,I) are segregated in ER subdomains displaying FAM134B at the limiting membrane and are eventually delivered to LAMP1-positive endolysosomal compartments for clearance (Fregno et al, 2018(Fregno et al, , 2021;;Rudinskiy and Molinari, 2023).If ERAD clients would co-opt the same pathway for lysosomal clearance upon ERAD inactivation, it is expected that NHK delivery to the LAMP1-positive compartment is substantially inhibited in cells lacking FAM134B.To test this hypothesis, the experiments described in the previous sections were reproduced in mouse embryonic fibroblasts (MEF) subjected to CRISPR/Cas9 genome editing to knockout FAM134B (Fig. 4A, lane 2).As shown above for other cell lines, also in wild-type (WT) MEF (Fig. 4B, upper panels, C) and in MEF lacking the FAM134B (Fig. 4B, lower panels, C) the ERAD client NHK is not delivered to  ).The back-transfection of V5-tagged FAM134B in FAM134B-KO MEF (Fig. 4A, lane 3) restores NHK delivery to the LAMP1-positive endolysosomes (Fig. 4F,K).The backtransfection of FAM134BLIR (Fig. 4A, lane 4), an inactive variant of the ER-phagy receptor that carries mutations in the LIR domain preventing engagement of cytosolic LC3 molecules (Fregno et al, 2018), is not restoring NHK delivery to endolysosomes (Fig. 4G,K).The FAM134B-driven deviation of NHK into the ERLAD pathways is also induced when ERAD of NHK is abolished upon MEF exposure to the proteasomal inhibitor PS341 (Fig. 4H-K).

Involvement of autophagy genes in compensatory ERLAD pathways
ER-phagy receptors control clearance of ER subdomains upon engagement of various autophagy gene products that will determine if ERLAD will proceed via macro-ER-phagy involving doublemembrane autophagosomes (as for misfolded procollagen) (Forrester et al, 2019), or via other types of ER-phagy that will not involve autophagosomes and their biogenesis (as for ATZ) (Chino and Mizushima, 2023;Fregno et al, 2018;Fregno et al, 2021;Reggiori and Molinari, 2022).Previous work in our lab showed that FAM134B-controlled delivery of ATZ polymers to LAMP1-positive endolysosomes involves the LC3 lipidation machinery but does not involve the autophagosome biogenesis machinery.Consistently, deletion of the autophagy gene Atg7 that abolishes LC3 lipidation (Komatsu et al, 2005) prevents ATZ delivery to endolysosomal compartments.In contrast, deletion of ATG13, a crucial component of the autophagosome biogenesis machinery (Hosokawa et al, 2009;Kaizuka and Mizushima, 2016;Suzuki et al, 2014) does not impact on ATZ delivery to the degradative district (Fregno et al, 2018).The repetition of these experiments to monitor the case of the ERAD client NHK confirms that the misfolded protein is not normally delivered to the LAMP1-positive endolysosomal compartment (Figs. 5A,, unless the ERAD pathway has been inactivated upon cell exposure to the α1,2-mannosidases inhibitor KIF or the proteasomal inhibitor PS341 (Figs. 5B,C,H and Figs. 2 and 4).In cells lacking ATG7, which show impaired generation of the lipidated form of LC3 (LC3-II, Fig. 5D), the delivery of NHK to the LAMP1-positive degradative compartment is abolished (Fig. 5E,H).In cells lacking ATG13 (Fig. 5F), the delivery of NHK to the LAMP1-positive degradative compartment proceeds unperturbed (Fig. 5G,H).These results recapitulate the phenotype previously observed for clearance of ATZ polymers, which is hampered in cells with defective LC3 lipidation (Chu et al, 2014;Fregno et al, 2018;Hidvegi et al, 2010;Kroeger et al, 2009;Pastore et al, 2013;Sun et al, 2023;Teckman and Perlmutter, 2000), but remains unaffected in cells with defective autophagosome biogenesis (Fregno et al, 2018).

Pharmacologic and genetic inhibition of ERAD triggers lysosomal delivery of BACE457Δ
Monitoring the fate of BACE457Δ, another folding-defective polypeptide that has been used extensively to characterized mechanistically the ERAD pathways (Bernasconi et al, 2010a;Bernasconi et al, 2010b;Cali et al, 2008;Eriksson et al, 2004;Horimoto et al, 2013;Molinari et al, 2003;Molinari et al, 2002;Ninagawa et al, 2011;Olivari et al, 2006;Olivari et al, 2005;Sokolowska et al, 2015), we confirmed that ERLAD acts as surrogate catabolic pathway to remove misfolded polypeptides from cells with dysfunctional ERAD (Fig. 6).As shown above for NHK, biochemical analyses of BACE457Δ decay confirm that the inhibition of lysosomal enzymes does not affect clearance from the ER (Molinari et al, 2002), unless cells have dysfunctional ERAD upon inhibition of cytosolic proteasomes with PS341 (Fig. 6A, lanes 6, 7 and gray zones in Fig. 6B) or of luminal mannosidases with KIF (Fig. 6A, lanes 10, 11 and gray zones in Fig. 6C).CLSM analyses confirm delivery of BACE457Δ within LAMP1-positive endolysosomes in cells with dysfunctional ERAD (Fig. 6D-G), and the involvement of FAM134B in the surrogate catabolic pathways activated under these circumstances (Fig. 6H-O).

Discussion
A stringent protein quality control operates in the ER of eukaryotic cells.
Proteins that have completed their folding program are released from ER-resident molecular chaperones and exit the ER to be delivered to their final intra-or extracellular destination.Folding is error-prone, and the rate of misfolding is substantially enhanced by mutations in the polypeptide sequence.The incapacity to fold correctly, results in selection of the aberrant gene product for degradation, or in the formation of aggregates that are retained in the biosynthetic compartment in soluble or insoluble forms.Dedicated machineries are available in the ER lumen and membranes to distinguish misfolded or incompletely folded polypeptides to be retained in the ER lumen, from native and functional proteins to be released (Ellgaard et al, 1999).Incompletely folded polypeptides retained in the ER are exposed to the folding environment and can eventually reach the native, transport-permissive architecture.
When folding is impossible, the polypeptides are actively deviated into ERAD pathways that ensure their transport into the cytosol for proteasomal degradation (Fig. 1A).An increasing number of misfolded proteins is emerging in the literature that cannot enter ERAD pathways.In many cases, these are large polypeptides or polypeptides that are prone to form aggregates or polymers.A variety of ERLAD pathways are available in nucleated cells to segregate ERAD-resistant polypeptides in dedicated ER subdomains and to deliver them to endolysosomal/ vacuolar degradative compartments (Fig. 1B) (Rudinskiy and Molinari, 2023).For ERAD clients, numerous studies show that clearance from the ER is delayed, and not abolished upon inactivation of the ubiquitin-proteasome system.This also applies for cytosolic misfolded proteins, whose clearance from cells hosting inactive proteasomes has been proposed to rely on intervention of the giant protease tripeptidyl peptidase II, which shows enhanced activity in proteasome-inhibitor adapted cell (Geier et al, 1999;Glas et al, 1998;Tomkinson, 2019).Our study demonstrates that the intervention of ERLAD pathways is not limited to clearance of large proteins that fail to be dislocated across the ER membrane for ERAD.Rather, ERLAD may be engaged by ERAD clients when their preferred road to destruction is dysfunctional or saturated by an excess of aberrant gene products (Fig. 7).

Cell Lines, transient transfections, pharmacologic inhibition
Flp-In TM -3T3 cells (Thermo Fisher) stably expressing ATZ-HA or NHK-HA were generated following manufacturer instructions and cultured in DMEM supplemented with 10% FCS and 150 μg/ml Hygromycin.MEF and HEK293 cells were grown in DMEM/10% FBS.HEK293FT cells expressing reduced levels of OS9.1 and OS9.2 are described in (Bernasconi et al, 2008).FAM134B-deficient MEF cells (CRISPR FAM134B) were generated using CRISPR/Cas9 genome editing protocol as described in (Fumagalli et al, 2016).Transient transfections were performed using JetPrime transfection reagent (PolyPlus) following manufacturer's protocol.BafA1 (Calbiochem) was used at 50 nM for 12 h; KIF (Toronto Research Chemicals) was used at 200 μM for 12 h; PS341 (LubioScience) was used 12 h at 100 nM for Flp-In TM -3T3 cells or at 5 nM for MEF cells.In wash/out experiments Flp-In TM -3T3 cells were incubated for 8 h with 100 nM BafA1 and indicated drugs.
To induce autophagy in MEF WT and ATG7-KO, cells were washed three times with Earle's balanced salt solution (EBSS, Thermo Fisher) and then incubated for 4 h with 100 nM BafA1.

Cell lysis, immunoprecipitation, and western blot
After treatments, cells were washed with ice-cold PBS containing 20 mM NEM and lysed with RIPA Buffer in HBS pH 7.4 supplemented with protease inhibitors.Post-nuclear supernatants (PNS) were collected after centrifugation at 10,600 × g for 10 min.For immunoprecipitations, PNSs were diluted with lysis buffer and incubated with Protein A (1:10 w/v, swollen in PBS) and select antibodies at 4 °C.After three washes of the immunoprecipitates with 0.5% Triton X-100 in HBS pH 7.4, beads were denatured for 5 min at 95 °C and subjected to SDS-PAGE.Proteins were transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad).Membranes were blocked with 10% (w/v) non-fat dry milk (Bio-Rad) in TBS-T and stained with primary antibodies diluted in TBS-T followed by HRP-conjugated secondary antibodies or Protein A diluted in TBS-T.Membranes were developed using Western Bright ECL or Quantum (Advansta), and signals captured on Fusion FX (Vilber).Images were quantified with the Evolution Capture Edge (Vilber).

Metabolic labeling
Seventeen hours after transient transfections, cells were pulsed with 0.05 mCi [35 S]methionine/cysteine mix and chased for the indicated time points with DMEM supplemented with 5 mM cold methionine and cysteine.Cells were detergent-solubilized and radiolabeled proteins were revealed with Typhoon FLA 9500, version 1.0 (GE Healthcare).Radioactive signals were quantified using the ImageQuant software (Molecular Dynamics, GE Healthcare).

Figure 1 .
Figure 1.Proteasomal and lysosomal pathways for clearance of misfolded proteins from the ER.
positive endolysosomes.Inactivation of the ERAD pathway with KIF promotes NHK delivery to the LAMP1-positive degradative compartments in WT MEF (Fig.4D,K), which is virtually abolished upon deletion of FAM134B (Fig.4E,K, pCDNA3

ProteasomeFigure 7 .
Figure 7. Schematic representation of how pharmacologic (blue) and genetic (red) ERAD perturbation triggers compensatory ERLAD programs.Defective proteasomal clearance of NHK and BACE457Δ activates FAM134Bdriven ERLAD.Other clients or maintenance of ER homeostasis in specific tissues may activate other ER-phagy receptors (e.g., CCPG1 in the pancreas and FAM134B-2 in the liver).